Large-scale Production of LGR5-positive Bipotential Human Liver Stem Cells.

The gap between patients on transplant waiting lists and available donor organs is steadily increasing. Human organoids derived from Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5)-positive adult stem cells represent an exciting new cell source for liver regeneration; however, culturing large numbers of organoids with current protocols is tedious and the level of hepatic differentiation is limited. Here, we established a new method for the expansion of large quantities of human liver organoids in spinner flasks. Due to improved oxygenation in the spinner flasks, organoids rapidly proliferated and reached an average 40-fold cell expansion after two weeks, compared to 6-fold expansion in static cultures. The organoids repopulated decellularized liver discs and formed liver-like tissue. After differentiation in spinner flasks, mature hepatocyte markers were highly upregulated compared to static organoid cultures, and cytochrome p450 activity reached levels equivalent to hepatocytes. CONCLUSION: We established a highly efficient method for culturing large numbers of LGR5-positive stem cells in the form of organoids, which paves the way for the application of organoids for tissue engineering and liver transplantation.